Elastin producing fibroblast formulations and methods of using the same

ABSTRACT

The present invention describes therapeutic compositions comprising fibroblasts that have been stimulated to increase expression of extracellular matrix components or elastin, or to produce enhanced elastogenesis or the appearance thereof at a site of administration. The therapeutic fibroblast formulations can be prepared using a variety of elastogenic agents, including digests of mammalian elastin, chemically digested plant extracts comprising elastin-like peptides, and synthetic elastogenic peptides. The invention further comprises cosmetic and pharmaceutical treatment methods using the therapeutic fibroblast compositions of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part U.S. Ser. No. 10/778,253filed Feb. 13, 2004 titled “ELASTIN DIGEST COMPOSITIONS AND METHODSUTILIZING SAME” and of U.S. Ser. No. 11/405,843, filed Apr. 17, 2006,and claims the benefit of U.S. provisional application Ser. No.60/681,600, filed May 17, 2005, all of which are herein incorporated byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable

JOINT RESEARCH AGREEMENT

N/A

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A CD

Not Applicable

BACKGROUND OF THE INVENTION

Elastin is an amorphous protein present in the elastic fibers of tissuessuch as arteries, blood vessels, skin, tendons and elastic ligaments,the abdominal wall, and lungs. Unlike other fibrous tissues likecollagen, elastin is unique in that it may be stretched to over 150percent of its original length, and can rapidly return to its originalsize and shape. This property of elastin provides tissues thatincorporate it the ability to resume their original form afterstretching due to, for example, blood flow, breathing, or bending. Likecollagen protein, elastin contains about 30% glycine amino acid residuesand is rich in proline. Elastin differs from collagen in that itcontains very little hydroxyproline or hydroxylysine. Elastin has a veryhigh content of alanine and also contains two unique amino acids,isodesmosine and desmosine. These unique amino acids, formed afteramalgamation of three or four lysines, are responsible for crosslinkingof adjacent tropoelastin molecules into the resilient elastin polymer,giving it the ability to return to its original shape after stretching.

Skin aging is a complex process determined by the genetic endowment ofthe individual as well as by environmental factors. In developedcountries, interest in cutaneous aging is in large part the result of aprogressive rise in absolute number and proportion of population who areelderly. Normal or intrinsic aging induces a progressive loss ofextracellular matrix (ECM), cellularity and elasticity of skin with age.Exposure of the skin to ultraviolet and visible light, numerouschemicals, as well as accumulation of calcium and certain metabolitesmay induce structural damage of the existing ECM, that eventually leadto loss of elasticity and formation of wrinkles as a result of localcollapse of the dermal tissue supporting epidermal layers. Especiallysevere and permanent loss of elasticity occurs after structural damageor enzymatic degradation of the elastic fibers (i.e. mid-dermalelastolysis), in certain metabolic diseases and after menopause. Geneticdiseases associated with a decrease in cutaneous elastic fibersadditionally lead to loss of elasticity, lax skin and premature wrinkleformation. Costello Syndrome, Cutis Laxa and Pseudoxanthoma Elasticum,for example, lead to premature aging most noticeably characterized bywrinkling and folding of the skin in children (pre-teenage) sufferingfrom these illnesses. Loss of elastin, in contrast to other ECMcomponents, cannot be spontaneously replaced by fully differentiatedfibroblasts residing in adult human skin. In fact, dermal fibroblastsare mostly in the quiescent state and can be only engaged in verylimited tissue remodeling and repair that includes neo-synthesis ofcollagens, glycoproteins and proteoglycans, but exclusively lacks ofelastogenesis.

While other ECM components provide the skin with mechanical strength andsecure its proper hydration, the network of elastic fibers is solelyresponsible for skin resiliency. Elastic fibers are composed of twomajor components: a scaffold of 10-12 nm microfibrils made up of severaldistinct glycoproteins and an amorphous core, consisting of elastin.Elastin polymer is formed after enzymatic cross-linking of the multiplemolecules of the 70-73 kDa precursor protein called tropoelastin.Tropoelastin (often referred as a soluble elastin) is synthesized bydermal fibroblasts and secreted in association with the 67 kDa elastinbinding protein (EBP) that acts as a molecular chaperone protecting thehighly hydrophobic tropoelastin molecules from intracellularself-aggregation and premature degradation and facilitating their properassembly on the microfibrillar scaffold in the extracellular space.Tropoelastin molecules are then polymerized into the insoluble elastinvia lysyl oxidase-dependent cross-linking of their lysines residues.Mature (insoluble) elastin, synthesized almost exclusively during lategestation and early childhood, is metabolically inert and remains themost durable element of extracellular matrix that may last over theentire human lifespan in undisturbed tissues. Although the primaryphysiological role of insoluble elastin is to serve as a structuralcomponent of elastic fibers, there is increasing evidence that bothelastin and its soluble degradation products can interact with the cellsurface elastin receptor and induce intracellular signals modulatingcellular proliferation, migration, and synthetic abilities.

SUMMARY OF THE INVENTION

The protein motif VGVAPG (SEQ ID NO. 1) has been previously shown tostimulate proliferation/migration of monocytes, dermal fibroblasts, andsmooth muscle cells through its interaction with the cell-surfaceelastin receptor. Other GXXPG (SEQ ID NO. 2) sequences recognized by BA4antibody are also known ligands for the elastin receptor. More recently,it has been shown that elastin peptides, liberated through proteolyticdigestion of bovine ligamentum nuchae and containing elastin receptorligand sequences (GXXPG) (SEQ ID NO. 2) also induce elastogenesis indermal fibroblasts through interaction with the elastin receptor.

We have developed a novel preparation consisting of an enzymaticdigestion of bovine ligamentum nuchae (ProK-60) that, in addition to themajor bulk of water soluble peptides derived from different domains ofelastin, contains minute admixtures of immuno-detected fragments ofmicrofibrillar and other elastic fiber-associated proteins as well asfragments of cytokines and growth factors. Results of in vitro studies,involving fibroblasts derived from skin of healthy Caucasian females ofdifferent ages (ranging from 3 to 61 years old), demonstrated that thispreparation can stimulate dermal fibroblast proliferation and inducedeposition of new extracellular matrix particularly rich of elasticfibers. Additional results indicated that ProK-60 induces production ofnew elastic fibers in organ cultures of skin explants derived frombiopsies of normal-looking abdominal skin of multiple females (28-55years old) with stretch marks. We demonstrate herein that human dermalfibroblasts pretreated in vitro with ProK-60 produced a dense network ofelastic fibers after their injection into the skin of athymic nude mice.

Accordingly, the present invention comprises a therapeutic compositionfor enhancing or stimulating elastogenesis or the appearance ofelastogenesis comprising a therapeutically effective amount of apopulation of cultured dermal fibroblasts, wherein the population ofcultured dermal fibroblasts is pretreated with an elastogeniccomposition. According to one embodiment of the invention, thepopulation of cultured dermal fibroblasts is derived from a skin biopsy.According to one embodiment, the skin biopsy comprises cells of thestratum basale layer.

According to an embodiment of the invention, the therapeutic compositioncomprises cultured dermal fibroblasts that have been pretreated with anelastogenic composition. An elastogenic composition according to theinvention may comprise, for example, one or more of ProK-60, E91,ProK-60P, a peptide having the sequence GXXPG (SEQ ID NO. 2), wherein Xrepresents any of the natural amino acids, or a variety of otherelastogenic peptides, including those described herein. In oneembodiment, the elastogenic composition used to prepare a therapeuticcomposition of the invention comprises peptides obtained or derived froma plant. In another embodiment, an elastogenic composition used toprepare a therapeutic composition according to the invention comprisespeptides obtained or derived from rice bran. The elastogenic peptide maybe, according to one embodiment, a synthetic peptide.

The present invention comprises compositions and methods for enhancingor stimulating elastogenesis, or providing the appearance of increasedelastogenesis, comprising administering a therapeutically effectiveamount of a composition of the invention. Accordingly, the inventionfurther comprises, according to one embodiment, a method for improvingappearance or elasticity of a tissue comprising administering to amammal in need thereof an effective amount of a composition comprising apopulation of cultured dermal fibroblasts, wherein the population ofcultured dermal fibroblasts is pretreated with an elastogeniccomposition. According to various embodiments, the cultured dermalfibroblasts are pretreated with an elastogenic composition comprisingone or more of ProK-60, E91, ProK-60P, a peptide having the sequenceGXXPG (SEQ ID NO. 2), wherein X represents any of the natural aminoacids, and one or more of a variety of other elastogenic peptides,including but not limited to those described herein. In one embodiment,the elastogenic composition used to prepare the therapeutic compositionsaccording to the invention comprises peptides obtained or derived from aplant. In another embodiment, the elastogenic composition comprisespeptides obtained or derived from rice bran.

The invention further comprises a method for stimulating new bloodvessel formation in a tissue comprising administering to a mammal inneed thereof an effective amount of a composition comprising apopulation of cultured dermal fibroblasts, wherein the population ofcultured dermal fibroblasts is pretreated with a composition comprisingan elastogenic compound.

A therapeutic composition of the invention may be administered by avariety of methods. According to one embodiment, a therapeuticcomposition of the invention is administered by injection.Administration of a therapeutic composition of the invention, accordingto one embodiment, stimulates elastogenesis at a site of administration.Administration of a therapeutic composition of the invention, accordingto one embodiment, improves the appearance of visible lines or wrinkles.Administration of a therapeutic composition of the invention, accordingto another embodiment, improves the appearance of scar tissue.

According to one embodiment of the invention, the therapeuticcomposition is administered at a site in preparation for plasticsurgery.

The therapeutic compositions of the invention may have cosmeticpurposes. The composition of the invention can be used, for example, toimprove the appearance of skin, such as by reduction or removal offacial lines and wrinkles, as well as reduction or removal of stretchmarks. According to one embodiment, the invention comprises a method ofimproving appearance of skin comprising applying a therapeuticcomposition of the invention to skin in an amount sufficient tostimulate elastogenesis. Moreover, the compositions of the invention maytighten loose, sagging skin on the face and other parts of the bodyincluding arms, legs, chest and neck areas, or give the appearance ofreducing wrinkles. Other methods of use of the compounds of the presentinvention include stimulation of smooth muscle cells and gingivalfibroblasts to produce elastin and fibrillin (oxytalan fibers),respectively, for the treatment of neointimal thickening and looseningof teeth (gingivitis), respectively. Accordingly to some embodiments,the invention comprises compositions and methods for stimulating dermalcell differentiation.

The compositions of the present invention may have other therapeuticpurposes. For example, the compositions of the present invention may beused to treat post infarct scar. According to this embodiment, theinvention includes a method of treating post infarct scar comprisingapplying a therapeutic composition of the invention to a post infarctscar in an amount sufficient to stimulate deposition of elastin at thepost infarct scar.

Furthermore, the therapeutic compositions of the invention may be usedto enhance wound healing and to prevent and treat cutaneous hypertrophicscars. Accordingly, another embodiment of the invention includes amethod of promoting wound healing and reducing scarring comprisingapplying a pretreated fibroblast composition of the invention to thewound in an amount sufficient to stimulate deposition of elastin at asite of injury.

According to one embodiment, the pretreated fibroblast formulations ofthe invention can be used to treat a site prior to tissue transplant.Certain surgical methods include a preliminary incision which isperformed in advance of a transplantation to stimulate new blood vesselgrowth at a site of future transplant. The present invention allows asurgeon to avoid such a preliminary incision step, by providing aninjectable pretreated fibroblast formulation. Injection of a pretreatedfibroblast formulation of the invention at such a site can, according toone embodiment, substitute for a preliminary incision by stimulating newblood vessel formation.

DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of necessary fee.

FIG. 1A: Representative Northern blots with elastin cDNA probeH-11-cultures of dermal fibroblast derived from 50-year-old female(upper panel) and results of statistical analysis of Northern blots ofthree different RNA samples from cultures derived from three differentpatients (lower panel) demonstrate that normal human fibroblastsincubated for 24 h in the presence of 25 μg/ml of ProK-60 hadsignificantly higher abundance of elastin mRNA, as compared to untreatedcontrol. The intensity of the elastin hybridization signal wasnormalized to the abundance of GAPDH in the corresponding blot and theresulting values are shown in the bar graph in arbitrary units.

FIG. 1B: Results of quantitative analysis (mean±S.D.) of a typicalexperiment using 3-day-long metabolic labeling of quadruplicate cultureswith radioactive [³H]-valine followed by biochemical isolation ofinsoluble elastin demonstrate that dermal fibroblasts derived from50-year-old female treated with ProK-60 and ProK-60P produced morelabeled insoluble elastin than the untreated controls. This stimulationwas dose dependent in the 5-25 μg/ml concentration range. Values ofmean±S.D. from five different experiments were collected and respectiveresults from ProK-60- and ProK-60P-treated cultures were statisticallycompared with untreated controls.

FIG. 1C: Representative photomicrographs of 10-day-old cultures ofdermal fibroblasts immunostained with anti-tropoelastin antibody.Fibroblasts derived from females of different ages (nucleicounterstained with red dye) treated either with ProK-60 or ProK-60Pproduced more elastic fibers (green fluorescence) than their untreatedcounterparts.

FIG. 2: Morphometric analysis of extracellular matrix componentsimmunostained with the respective specific antibodies in 10-day-oldcultures of dermal fibroblasts derived from females of different ages.

FIG. 3: Results of typical experiment aimed at quantitative assessmentsof cellular proliferation of dermal fibroblasts derived from 50-year-oldfemale and cultured for 3 days in the presence and absence of 25 μg/mlof ProK-60 and ProK-60P.

FIG. 4: Results of experiments demonstrating that addition of lactose(reagent inactivating the cell surface elastin receptor) to cultures ofhuman skin fibroblasts simultaneously treated with ProK-60 causedsignificant inhibition of the proliferative and elastogenic effects ofProK-60. Addition of fucose, a sugar that does not interfere withelastin receptor, did not diminish beneficial effect of ProK-60. Results(mean±S.D.) from three different experiments were combined andstatistically evaluated.

FIG. 5. Representative micrographs of the Movat pentachrome-stainedtransverse sections of skin biopsy explants derived from abdominal skinof two, 37- and 44-year-old, female patients. Explants were maintainedin organ cultures for 10 days in the presence and absence of 25 μg/ml ofProK-60. The results indicate that ProK-60 was able to penetrate intothe cultured explants and induce production of new elastic fibers.Movat's pentachrome stain shows elastin as black, collagen as yellow,cells red and nuclei as dark blue (original magnification 200×).

FIG. 6. Results of quantitative morphometric analysis detecting elasticfibers in dermal explants (deriving from five female patients) culturedfor 10 days in the presence and absence of ProK-60.

FIG. 7. Representative micrographs of transverse sections of skin-biopsyexplants derived from abdominal skin of a 34-year-old female patient.Explants were maintained in organ cultures for 10 days in the presenceand absence of 25 μg/ml of ProK-60. Immuno-peroxidase detection (brown)of proliferative antigen, PCNA, indicates that ProK-60 mostly stimulatedproliferation and migration of cells located in the stratum basale intothe superficial dermis. Movat's pentachrome stain demonstrated thatfibroblasts located near the dermo-epidermal junction produced moreelastic fibers (black) in ProK-60-treated explants than in controlcounterparts.

FIG. 8. Representative photomicrographs comparing transverse sections ofskin from the athymic nude mice injected with dermal fibroblasts derivedfrom 50-year-old female. In contrast to skin of control mice injectedwith vehicle that demonstrate only few elastic fibers, all one-, three-and four week-old implants contain injected human skin fibroblasts,which produced a new extracellular matrix, rich of elastic fibers.Injected fibroblast that were pre-incubated with ProK-60 produced moreelastic fibers than their untreated counterparts.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,“about 50%” means in the range of 45%-55%.

The term “cosmetic,” as used herein, refers to a beautifying substanceor preparation which preserves, restores, bestows, simulates, orenhances the appearance of bodily beauty or appears to enhance thebeauty or youthfulness, specifically as it relates to the appearance oftissue or skin.

The term “improves” is used to convey that the present invention changeseither the appearance, form, characteristics and/or the physicalattributes of the tissue to which it is being provided, applied oradministered. The change in form may be demonstrated by any of thefollowing alone or in combination: enhanced appearance of the skin;increased softness of the skin; increased turgor of the skin; increasedtexture of the skin; increased elasticity of the skin; decreased wrinkleformation and increased endogenous elastin production in the skin,increased firmness and resiliency of the skin.

The terms “mimetic,” “peptide mimetic” and “peptidomimetic” are usedinterchangeably herein, and generally refer to a peptide, partialpeptide or non-peptide molecule that mimics the tertiary bindingstructure or activity of a selected native peptide or protein functionaldomain (e.g., binding motif or active site). These peptide mimeticsinclude recombinantly or chemically modified peptides, as well asnon-peptide agents such as small molecule drug mimetics, as furtherdescribed below.

The term “dimer”, as in a peptide “dimer”, refers to a compound in whichtwo peptide chains are linked; generally, although not necessarily, thetwo peptide chains will be identical and are linked through a linkingmoiety covalently bound to the terminus of each chain.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, rash, or gastric upset.In a preferred embodiment, a pharmaceutical composition of the inventionis not immunogenic when administered to a human patient for therapeuticpurposes.

“Providing” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. Thus, as used herein, theterm “providing” can include, but is not limited to, providingcompositions into or onto the target tissue; providing compositionssystemically to a patient by, e.g., intravenous injection whereby thetherapeutic reaches the target tissue.

Unless otherwise indicated, the term “skin” means that outer integumentor covering of the body, consisting of the dermis and the epidermis andresting upon subcutaneous tissue.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. In part, embodiments of the present invention are directedto improve the functionality, the appearance, the elasticity, and/or theelastin content of mammalian tissue. As it applies to skin, it ismeasured by elasticity, turgor, tone, appearance, degree of wrinkles,and youthfulness. As it applies to smooth muscle cells, blood vessels,it is measured by increased elasticity (elastin/elastic fiber synthesisand deposition) and decreased neointimal thickening (smooth muscle cellproliferation). The methods herein for use contemplate prophylactic useas well as curative use in therapy of an existing condition. Therapeuticcompositions of the invention may comprise cosmetic preparations orpharmaceutical formulations.

The terms “therapeutically effective” or “effective”, as used herein,may be used interchangeably and refer to an amount of a therapeuticcomposition of the present invention. For example, a therapeuticallyeffective amount of a composition of the invention is a predeterminedamount calculated to achieve the desired effect, i.e., to effectivelypromote elastin production, collagen production, cell proliferation, orimproved appearance, or improved tissue elasticity in an individual towhom the composition is administered. The tissue in need of suchtherapeutic treatment may, for example, present lines or wrinkles, sundamaged tissue, or scar tissue.

The term “tissue” refers to any aggregation of similarly specializedcells which are united in the performance of a particular function. Asused herein, “tissue”, unless otherwise indicated, refers to tissuewhich includes elastin as part of its necessary structure and/orfunction. For example, connective tissue which is made up of, amongother things, collagen fibrils and elastin fibrils satisfies thedefinition of “tissue” as used herein. Additionally, elastin appears tobe involved in the proper function of blood vessels, veins, and arteriesin their inherent visco-elasticity. Thus, “tissue” thus includes, but isnot limited to skin fibroblasts and smooth muscle cells including humanaortic smooth muscle cells.

The term “unit dose” when used in reference to a therapeutic compositionof the present invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,excipient, carrier, or vehicle.

For simplicity and illustrative purposes, the principles of theinvention are described by referring mainly to an embodiment thereof. Inaddition, in the following descriptions, numerous specific details areset forth in order to provide a thorough understanding of the invention.It will be apparent however, to one of ordinary skill in the art, thatthe invention may be practiced without limitation to these specificdetails.

Preparation of Pretreated Fibroblasts

One embodiment of the present invention includes a pretreated fibroblastformulation prepared by cell culture of patient dermal fibroblasts. Inanother embodiment, the formulation may be re-introduced into a patientin need of a stimulatory (e.g., elastogenic) cosmetic or therapeuticeffect.

According to one embodiment, the present invention comprises therapeuticpreparations comprising fibroblasts that have been stimulated, e.g., toincrease expression of elastin, by contacting such fibroblasts withstimulatory compositions as described herein. The invention furthercomprises methods of using such pretreated fibroblast formulations intherapeutic (including for example cosmetic) applications. According toone embodiment, the invention comprises methods of using autologousfibroblasts obtained from an individual patient and pretreated in vitrowith ProK-60 and other stimulatory formulations comprising injectingsuch pretreated fibroblasts back into the same patient for therapeutic(including for example cosmetic) reasons.

According to one embodiment, the invention comprises therapeuticpreparations or formulations comprising pretreated fibroblasts.Pretreated fibroblasts of the invention are prepared, according to oneembodiment, by obtaining a skin biopsy of a patient, culturing andpassaging the skin biopsy to obtain a population of fibroblasts from thebiopsy, treating the cultured fibroblasts with a stimulatory compositionsuch that, for example, one or more fibroblasts of the population arestimulated to increase production of extracellular matrix, to increaseelastogenesis, or to enhance or improve the appearance of tissue at thesite of injection (an “elastogenic” composition), and washing thetreated fibroblasts to provide a population of pretreated fibroblastssuitable for injection into a site of a patient. According to oneembodiment, the washing step may be omitted in the preparation of suchpretreated fibroblasts. According to another embodiment, the pretreatedfibroblasts may be further combined with an elastogenic compositionprior to administration.

The elastogenic composition used to stimulate the population offibroblasts may comprise one or more of a variety of peptides, which mayinclude for example a mammalian-derived elastin digest, a plant-derivedextract comprising an elastin-like peptide, or a synthetic elastogenicpeptide. In one embodiment, the stimulatory composition is derived fromligamentum nuchae. In another embodiment, the stimulatory composition isderived from a plant, for example, rice bran.

A tissue biopsy of about 2×2 mm in size can provide a primary culture ofat least 10 million fibroblasts. The fibroblast population can beincreased by further passaging (up to 2 times) according to knownmethods.

Various tissues in a mammal may suffer from a condition or state whereloss of elastin has occurred, where the existing elastin present in thetissue has lost its elasticity, or where the endogenous production ofelastin or tropoelastin in the tissue is inadequate. Such tissue is inneed of elastin as may be identified by a loss of tissue elasticity,reduced capacity or loss of required tissue elastic function, loss ofappearance or suppleness, or loss of tone. Once identified, such tissuemay be treated with the compositions of the invention.

Bovine Elastin Digests

Pretreated fibroblasts included in therapeutic formulations of thepresent invention may be prepared by contacting fibroblasts with anelastin digest. For example, commercially available, Elastin E91preparation from Protein Preparations, Inc., St. Louis, Mo., is asuitable elastin product to subject to digestion, having about 1,000 to60,000 dalton molecular weight. Additionally, a series of digestsavailable under the trade name ProK, and specifically ProK-60 andProK-60P, are elastin peptide mixtures derived from the proteolyticdigestion of insoluble elastin derived from bovine neck ligaments(commercially available from Human Matrix Sciences, LLC). The digestionis accomplished with Proteinase K enzyme. The commercially availableproducts will be referred to as E91 and ProK respectively.

Pretreated fibroblasts included in therapeutic compositions of thepresent invention may be prepared by contacting cultured fibroblastswith a composition comprising an elastogenic compound, for example, oneor more peptides derived or made from digests of elastin and/or collagencomprising tissue. Exemplary peptides are listed in Tables 1 and 2. Thepeptides may be synthetic. The therapeutic compositions of the inventionmay be cosmetic, or pharmacological and are useful for treatingmammalian tissue.

Fibrous protein tissue comprising elastin or collagen-like tertiarystructures and tropoelastin are examples of proteins and peptides whichmay be digested to produce elastin digests for preparing pretreatedfibroblasts of the present invention. Protein, peptides, elastin ortropoelastin may be obtained from various animal tissues, or fromplants, as described in U.S. Ser. No. Ser. No. 10/778,253, filed Feb.13, 2004 and U.S. application Ser. No. 11/405,843 filed Apr. 17, 2006,both of which are hereby incorporated by reference.

According to some embodiments, the elastin digests used to pretreatfibroblasts according the present invention comprise an amino acidsequence listed in Table 1.

TABLE 1 SEQ ID NO. Peptide Name 3 GAAPGGlycine-Alanine-Alanine-Proline-Glycine 4 GVVPGGlycine-Valine-Valine-Proline-Glycine 5 GGGPGGlycine-Glycine-Glycine-Proline-Glycine 6 GLLPGGlycine-Leucine-Leucine-Proline-Glycine 7 GIIPGGlycine-Isoleucine-Isoleucine-Proline- Glycine 8 GSSPGGlycine-Serine-Serine-Proline-Glycine 9 GTTPGGlycine-Threonine-Threonine-Glycine 10 GCCPGGlycine-Cysteine-Cysteine-Proline-Glycine 11 GMMPGGlycine-Methionine-Methionine-Proline- Glycine 12 GFFPGGlycine-Phenylalanine-Phenylalanine- Proline-Glycine 13 GYYPGGlycine-Tyrosine-Tyrosine-Proline-Glycine 14 GWWPGGlycine-Tryptophan-Tryptophan-Proline- Glycine 15 GDDPG Glycine-AsparticAcid-Aspartic Acid-Proline- Glycine 16 GNNPGGlycine-Asparagine-Asparagine-Proline- Glycine 17 GEEPG Glycine-GlutamicAcid-Glutamic Acid- Proline-Glycine 18 GQQPGGlycine-Glutamine-Glutamine-Proline- Glycine 19 GRRPGGlycine-Arginine-Arginine-Proline-Glycine 20 GHHPGGlycine-Histidine-Histidine-Proline-Glycine 21 GKKPGGlycine-Lysine-Lysine-Proline-Glycine 22 GPPPGGlycine-Proline-Proline-Proline-Glycine 23 G3Hyp3HypPGGlycine-3-hydroxyproline-3-hydroxyproline- Proline-Glycine 24G4Hyp4HypPG Glycine-4-hydroxyproline-4-hydroxyproline- Proline-Glycine25 RRPEV Arginine-Arginine-Proline-Glutamic Acid- Valine 26 QPSQPGGVGlutamine-Proline-Serine-Glutamine-Proline- Glycine-Glycine-Valine 27PGGV Proline-Glycine-Glycine-Valine 28 GPGVGlycine-Proline-Glycine-Valine 29 KPGV Lysine-Proline-Glycine-Valine 30GPGL Glycine-Proline-Glycine-Leucine 31 EGSA GlutamicAcid-Glycine-Serine-Alanine 32 PGGFProline-Glycine-Glycine-Phenylalanine 33 GGGAGlycine-Glycine-Glycine-Alanine 34 KPGKVLysine-Proline-Glycine-Lysine-Valine 35 PGGVProline-Glycine-Glycine-Valine 36 KPKA Lysine-Proline-Lysine-Alanine 37GPGGV Glycine-Proline-Glycine-Glycine-Valine 38 GPQAGlycine-Proline-Glutamine-Alanine 39 GGPGIGlycine-Glycine-Proline-Glycine-Isoleucine 40 PGPGAProline-Glycine-Proline-Glycine-Alanine 41 GPGGVGlycine-Proline-Glycine-Glycine-Valine 42 GQPFGlycine-Glutamine-Proline-Phenylalanine 43 GGKPPKPFGlycine-Glycine-Lysine-Proline-Proline- Lysine-Proline-Phenylalanine 44GGQQPGL Glycine-Glycine-Glutamine-Glutamine- Proline-Glycine-Leucine 45MRSL Methionine-Arginine-Serine-Leucine 46 GGPGIGlycine-Glycine-Proline-Gycline-Isoleucine

Elastin digests used for pretreating fibroblasts included in therapeuticcompositions of the invention may comprise one or more di-peptides.Suitable di-peptides found in the digests are listed in Table 2.

TABLE 2 Di-peptide Name CI Cysteine-Isoleucine GL Glycine-Leucine GAGlycine-Alanine KA Lysine-Alanine ST Serine-Threonine RFArginine-Phenylalanine PT Proline-Threonine QV Glutamine-Valine GIGlycine-Isoleucine PL Proline-Leucine GY Glycine-Tyrosine PIProline-Isoleucine KA Lysine-Alanine PA Proline-Alanine PYProline-Tyrosine KT Lysine-Tyrosine GF Glycine-Phenylalanine PTProline-Threonine KL Lysine-Leucine GV Glycine-Valine KILysine-Isoleucine QF Glutamine-Phenylalanine RA Arginine-Alanine CLCysteine-Leucine

Pretreated fibroblasts included in therapeutic compositions of theinvention may be prepared by contacting fibroblasts with an elastindigest comprising one or more peptides of the formulaGly-Xaa-Xbb-Pro-Gly (SEQ ID NO. 2) wherein Xaa and Xbb are any one ofthe 20 standard amino acids, 3-hydroxyproline, and 4-hydroxyproline, ortherapeutically acceptable acid addition salts thereof. In thesepeptides, the amino acids Xaa and Xbb may be the same or different aminoacids. Compositions comprising such peptides may be prepared by reactionof elastin comprising tissue with a digesting composition comprising,for example, proteinase K.

Pretreated fibroblast compositions of the invention may, according toother embodiments, be prepared with one or more elastogenic peptidescomprising the sequence PGGVLPG (SEQ ID NO. 47), VGVVPG (SEQ ID NO. 48),or IGLGPGGV (SEQ ID NO. 49).

Other Elastogenic Peptides, or Peptide Mimetics Thereof

In accordance with one embodiment of the present invention, fibroblastsare pretreated using novel peptides obtained from, derived from or basedon protein sequences found in plants, as described in U.S. Ser. No.11/405,843, filed Apr. 17, 2006 which is hereby incorporated byreference in its entirety.

In preferred embodiments, pretreated fibroblasts used in therapeuticformulations of the invention are prepared by contacting fibroblastswith synthetic peptides. Such synthetic peptides include peptidescomprising the sequence X₁GX₂X₃PG (SEQ ID NO. 50), wherein X₁ is V or I,X₂ is A, V or L, and X₃ is M, A or S. In one embodiment, a syntheticpeptide used to prepare pretreated fibroblasts comprises VGAMPG (SEQ IDNO. 51), VGLSPG (SEQ ID NO. 52), VGVAPG (SEQ ID NO. 53), IGAMPG (SEQ IDNO. 54), IGVAPG (SEQ ID NO. 55), or IGLSPG (SEQ ID NO. 56). In anotherembodiment, a synthetic peptide used to prepare pretreated fibroblastsis a peptide dimer comprising one or more peptide sequences identifiedherein.

In another embodiment, a synthetic peptide used to prepare pretreatedfibroblasts used in therapeutic formulations of the invention comprisesthe amino acid sequence X₁GX₂X₃PG, —X₄—X₁GX₂X₃PG (SEQ ID NO. 57),wherein -X₄- is a linking moiety; X₁ is independently selected from Vand I; X₂ is independently selected from A, V and L; and X₃ isindependently selected from M, A and S. The linking moiety can be anymoiety recognized by those skilled in the art as suitable for joiningpeptides so long as the compound retains the ability to interact with anelastin receptor and induce elastogenesis. The linking moiety may becomprised of, for example, but not limited to, at least one of alanineor any other amino acid, a disulfide bond, a carbonyl moiety, ahydrocarbon moiety optionally substituted at one or more availablecarbon atoms with a lower alkyl substituent. Optimally, the linkingmoiety is a lysine residue or lysine amide, i.e., a lysine residuewherein the carboxyl group has been converted to an amide moiety —CONH.

In one embodiment, a synthetic peptide used to prepare pretreatedfibroblasts used in a therapeutic composition of the invention comprisesVGAMPGAAAAAVGAMPG (SEQ ID NO. 58), VGLSPGAAAAAVGLSPG (SEQ ID NO. 59),VGVAPGAAAAAVGVAPG (SEQ ID NO. 60), IGAMPGAAAAAIGAMPG (SEQ ID NO. 61),IGVAPGAAAAAIGVAPG (SEQ ID NO. 62), or IGLSPGAAAAAIGLSPG (SEQ ID NO. 63).

Chemically Digested Plant Extracts

According to one embodiment, pretreated fibroblasts are prepared bycontacting cultured fibroblasts with chemically digested plant extracts.In one embodiment, such plant extracts are obtained from rice bran.Chemically digested rice bran extracts, the preparation of which isdescribed in more detail in U.S. Ser. No. 11/405,843, filed Apr. 17,2006, were found to be immuno-reactive with a panel of antibodies raisedto human tropoelastin. Furthermore, chemical digests of both soluble andinsoluble rice bran contained the unique crosslinking amino acid,desmosine. These characteristics suggest the presence of one or moreelastin-like peptides in rice bran. Thus, the pretreated fibroblasts ofthe present invention may be prepared using compositions comprising suchelastin-like peptide preparations.

Therapeutic Formulations

Therapeutic formulations, preparations, or compositions of the inventionmay comprise a cosmetic or pharmaceutical formulation, preparation, orcomposition. The preparation of such therapeutic compositions is wellunderstood in the art. Typically such compositions, if desired, may beprepared as sterile compositions either as liquid solutions orsuspensions, aqueous or non-aqueous, however, suspensions in liquidprior to use can also be prepared.

Therapeutic formulations comprising pretreated fibroblasts according tothe invention may be administered by a variety of methods known in theart. For example, therapeutic pretreated fibroblast formulations may beadministered topically, locally, perilesionally, perineuronally,intracranially, intravenously, intrathecally, intramuscularly,subcutaneously, intracavity, transdermally, dermally, or via animplanted device, and they may also be delivered by peristaltic means.According to a preferred embodiment, the therapeutic pretreatedfibroblast formulations of the invention are provided as injectableformulations. A therapeutic pretreated fibroblast formulation of theinvention may be administered parenterally by injection or by gradualinfusion over time.

A therapeutic pretreated fibroblast formulation of the invention maycomprise excipients which are pharmaceutically acceptable and compatiblewith the active ingredient and in amounts suitable for use in thetherapeutic methods described herein. Various excipients may be used ascarriers for compositions of the present invention as would be known tothose skilled in the art. For example, therapeutic pretreated fibroblastformulations of the invention may comprise an intravenous and salinecomprising mixture, dextrose, glycerol, ethanol or the like andcombinations thereof. In addition, if desired, a therapeutic compositionof the invention can contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents and the like whichenhance the effectiveness of the active ingredient. Exemplary of liquidcarriers are sterile aqueous solutions that contain no materials inaddition to the active ingredients and water, or contain a buffer suchas sodium phosphate at physiological pH value, physiological saline orboth, such as phosphate-buffered saline and Tris-HCl buffer. Stillfurther, aqueous carriers can contain more than one buffer salt, as wellas salts such as sodium and potassium chlorides, dextrose, propyleneglycol, polyethylene glycol and other solutes.

Additionally, in another embodiment of the invention, therapeuticpretreated fibroblast formulations of the invention comprise chemicalpreservatives, such as cetylpyridinium chloride, K-Sorbate, Na-Benzoate,various parabens, and/or other chemical preservatives.

Manganese salts (MnCl₂, MnSO₄ and MnaPCA) and trivalent iron (FerricAmmonium Citrate, FAC) have each been shown to individually stimulatethe production and assembly of new tropoelastin into new elastic fibers.The compositions of the present invention may be formulated to furtherinclude a manganese component and/or a trivalent iron component.Additionally, compounds comprising sodium are suitable additives fortherapeutic compositions of the present invention. Sodium has beenlinked to the stimulation of elastogenesis. Copper, an activator oflysyl oxidase (an enzyme that crosslinks tropoelastin molecules intoinsoluble polymeric elastin) is another suitable additive used in thetherapeutic compositions of the present invention.

Optionally, a manganese component may be added to a therapeuticcomposition of the invention. The manganese may be any manganesecompound, or a pharmaceutically acceptable salt thereof, but preferablyis MnCl₁, MnSO₄ and/or MnPCA, wherein the manganese component istypically present in an amount from about 0.5 to 10 weight percent,preferably from about 1 to 8 weight percent and most preferably fromabout 5 to 7 weight percent, wherein the manganese is present in anamount from about 5 to 20 weight percent of a complex. According to oneembodiment, the concentration of MnCl₂ in an injectable therapeuticcomposition of the invention is between 1 and 10 μM. According toanother embodiment, the concentrations of MnCl₂ in a topical therapeuticcomposition of the invention is between 5 and 50 μM.

Optionally a trivalent iron component (such as, but not limited to,Ferric Ammonium Chloride (FAC) may also be included in a therapeuticcomposition of the invention. The trivalent iron component stimulatesnew elastogenesis and assists in treatment of elastic tissue defects.The trivalent iron, when included in the composition, is generallypresent in an amount from about 5 to 20 weight percent. In oneembodiment the trivalent iron component is generally present in anamount from about 0.01 to 5 weight percent, preferably from about 0.02to 3 weight percent, and more preferably from about 0.03 to 2 weightpercent of the composition. In one embodiment, the concentration of FACincluded in an injectable therapeutic composition of the invention is5-50 μM. The concentration of FAC in a topical formulation can bebetween 25 and 250 μM.

Optionally, a sodium component, or pharmaceutically acceptable saltthereof, may also be included in a therapeutic composition of theinvention. The sodium component is generally present in about 5 to 20percent of the complex. The sodium component may generally be present inan amount of about 1 to 10 percent weight percent, or from about 5 to 7percent weight of the composition. In one embodiment, NaCl is providedin an injectable therapeutic composition of the invention at 160-170 μM.In another embodiment, NaCl is provided in a topical therapeuticcomposition of the invention at 800-850 μM.

A copper component may also be included in a therapeutic composition ofthe invention, and may be any copper compound or pharmaceuticallyacceptable salt thereof. The copper component inhibits elastase andassists in the treatment of elastic tissue defects. The copper compoundmay be in the form of copper sebacate. When included in a compositionthe copper component is generally present in an amount of about 5 to 20weight percent of the copper compound, such as copper sebacate. Thecopper component is generally present in an amount of about 0.01 to 5percent weight or from about 0.03 to 2 percent weight of thecomposition.

The pretreated fibroblasts included in the therapeutic formulations ofthe present invention may be prepared using the peptides describedherein as well as such peptides fused or chemically bonded to asubstrate by the method and materials disclosed in U.S. Pat. No.6,372,228, the entirety of which is hereby incorporated herein byreference.

Additives which aid in improving the elasticity of elastin comprisingtissues such as Tretinoin, vitamin E, sources of copper, zinc, and/ormagnesium ions, Retinol, copper peptides, and any one of the 20 standardamino acids may also be added to the therapeutic pretreated fibroblastcompositions of the present invention. Additives which induce depositionof tropoelastin on microfibril scaffolds, and compounds which inducelysyl oxidase activity, such as transforming growth factor beta-1, mayalso be added to such therapeutic compositions. Therapeutic compositionsof the present invention may include a therapeutically and biologicallycompatible excipient.

In another embodiment of the invention, therapeutic compositions of theinvention comprise other additives, such as hyaluronic acid. In anotherembodiment of the present invention, a method of clinical treatment forthe improvement of facial lines and wrinkles through injection of ahyaluronic acid/pretreated fibroblast compositions of the invention intosites presenting visible lines and wrinkles is provided. In suchinjections, the hyaluronic acid will act as a resorbable scaffold fordermal fibroblasts infiltration.

The pretreated fibroblast formulations of the invention can, accordingto one embodiment, comprise an elastogenic peptide or elastin extract ordigest, for example, any of the mammalian, plant or synthetic peptidesor extracts described herein, or any other elastogenic composition.Thus, for example, therapeutic pretreated fibroblast formulations of theinvention may further comprise mammalian elastin digests, prepared asdescribed for example in U.S. Ser. No. 10/778,253, filed Feb. 13, 2004,or plant digests comprising an elastin-like peptide or chemicallydigested rice bran extracts, both prepared as described for example inU.S. Ser. No. 11/405,843, filed Apr. 17, 2006, or synthetic peptidescomprising any of the peptide sequences discussed herein or peptidemimetics thereof. The therapeutic formulations of the inventionoptionally can include other components, including other epitopes forextracellular matrix proteins, cytokines and growth factors. Theseadditional components may include, for example, tropoelastin, thepeptide VGVAPG (SEQ ID NO. 1), desmosine, tropo-Exon 36, fibrillin 1,MAGP 1, LTBP2, versican, collagen type I, collagen type IV, fibronectin,EBP, PDGF, bFGF, FGF, and IL-1B.

A therapeutic composition of the invention optionally can include otheringredients, such as, but not limited to, anti-inflammatory agents,sunscreens/sunblocks, stimulators of protein synthesis, cell membranestabilizing agents (i.e., carnitine), moisturizing agents, coloringagents, opacifying agents and the like.

Additional components of the therapeutic compositions of the inventionmay include any suitable additive that has been used in cosmetics orother skin care compositions. These include, but are not limited to aloevera, antioxidants, azulene, beeswax, benzoic acid, beta-carotene, butylstearate, camphor, castor oil, chamomile, cinnamate, clay, cocoa butter,coconut oil, cucumber, dihydroxyacetone (DHA), elastin, estrogen,ginseng, glutamic acid, glycerin, glycolic acid, humectant,hydroquinone, lanolin, lemon, liposomes, mineral oil, monobenzone,nucleic acids, oatmeal, paba, panthenol, petroleum jelly, propeleneglycol, royal jelly, seaweed, silica, sodium lauryl sulfate sulfur,witch hazel, zinc, zinc oxide, copper, hyaluronic acid and shea butter.

Dosage Amounts

The dosage ranges for the administering of a therapeutic composition ofthe invention are those large enough to produce the desired effect inwhich the condition to be treated is ameliorated. The dosage should notbe so large as to cause adverse side effects. Generally, the dosage willvary with the age, condition, and sex of the patient, and the extent ofthe disease in the patient, and can be determined by one of skill in theart. The dosage can be adjusted in the event of any complication. Thetherapeutic formulation of the invention comprising pretreatedfibroblasts can be administered such that between about 10,000 and50,000 pretreated fibroblasts are provided at a given treatment site.According to one embodiment, a therapeutic composition of the inventionis provided in a volume of 100-300 μl, more preferably about 200 μl. Atherapeutic formulation of the invention has, in some embodiments,between about 50 and 250 pretreated fibroblasts/μl. In a preferredembodiment, a therapeutic formulation of the invention comprises betweenabout 100 and 200 pretreated fibroblasts/μl.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. A therapeuticamount of a therapeutic composition of the invention is an amountsufficient to produce the desired result, and can vary widely dependingupon the disease condition and the potency of the therapeuticformulation. In the present invention the desired result is, accordingto some embodiments, an improvement in elasticity of the tissue asdetermined by an improvement in the elastin content of the tissue,improved capacity and function of the tissue, or improved appearance,suppleness, and/or tone of the tissue being treated.

In general, routine experimentation will determine specific ranges foroptimal therapeutic effect for each composition and each administrativeprotocol, and administration to specific individuals will be adjusted towithin effective and safe ranges depending on the condition andresponsiveness of the individual to initial administrations.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as direct topical application, application via atransdermal patch and the like.

Heating of a site on a patient comprising tissue is known to open pores,activate the various mechanisms of a cell, and increase diffusion intosaid tissue and cells. Heating in connection with providing atherapeutic composition of the invention to a site comprising connectivetissue is therefore a preferred embodiment of the present invention.According to one embodiment, administration of a therapeutic compositionof the invention is combined with local heating at 39-41° C.

Methods of Use

According to some embodiments, a therapeutic composition of the presentinvention may be used to improve the elasticity, cell proliferation,endogenous elastin production, function, and/or appearance of propertiesof tissues by providing a source of new elastin directly to the site ofapplication. According to other embodiments, a therapeutic compositionof the invention may be used to induce dermal cell differentiation. Theinvention may be applied to tissue in a therapeutically effective amountfor the treatment of various diseases.

Compositions of the invention may provide the appearance of increasedelastogenesis in a tissue. One embodiment of the present inventionincludes therapeutic or cosmetic preparations comprising pretreatedfibroblasts useful in improving or enhancing the appearance of skin.Such therapeutic compositions are used, according to one embodiment, forthe restoration of cutaneous connective tissue proteins in the skin.Therapeutic compositions of the invention may be used, according toanother embodiment, to aid or facilitate the assembly of new elasticfibers in skin.

Further embodiments include methods of treating premature aging,including wrinkling and folding of the skin by administeringtherapeutically effective amounts of a composition of the invention.Embodiments further include methods of treating elastin or geneticabnormalities affecting elastic fibers in skin, including, but notlimited to, Costello Syndrome, Cutis Laxa and Pseudoxanthoma Elasticum,by administering therapeutically effective amounts of a composition ofthe present invention. The invention further includes uses oftherapeutic compositions of the invention in the treatment ofcardiovascular disorders that may benefit from stimulated elastogenesis.For example, the invention comprises methods of using a composition ofthe invention to stimulate elastic fiber formation in the scars formedafter heart infarcts.

A variety of useful compositions and formats, including bioabsorbablematerials or matrices may be used in conjunction with a therapeuticcomposition of the invention to treat a tissue requiring elastin.Further applications of therapeutic compositions of the inventioninclude to treat oral applications, e.g., treatment of minor ulcerationson gums or mouth tissue, to strengthen elastic fibers around follicles,e.g., to prevent hair loss, or to treat opthalmologic injuries orconditions, such as corneal ulcerations.

In another embodiment of the invention, a method of inhibiting theproduction of chondroitin sulfate-containing glycosaminoglycans isprovided. Glycosaminoglycans interfere with elastic fiber assembly.Therefore, inhibiting such production may be helpful, according to oneembodiment to decrease solar elastosis, for example, in treatingsun-damaged skin. The method comprises treating a site in need thereofwith a therapeutic formulation of the invention. Such inhibition ofchondroitin sulfate expression or synthesis aids in the deposition ofnewly synthesized elastin.

Many methods have been proposed and tested to promote wound healing andlimit scarring; however, better methods and compositions are stillneeded. Wounds that can be treated with the therapeutic compositions ofthe invention include, but are not limited to, cutaneous wounds, cornealwounds, and injuries to the epithelial-lined hollow organs of the bodyand post-infarct heart. Wounds suitable for treatment include thoseresulting from trauma such as burns, abrasions and cuts, decubitus andnon-healing varicose and diabetic ulcers, as well as wounds resultingfrom surgical procedures such as incisions and skin grafting. Accordingto one embodiment, a therapeutic composition of the invention can beused to treat infected wounds and ulcers.

Elastin production at a therapeutic site by a therapeutic composition ofthe invention will aid in promoting wound healing while limitingscarring. Initially, the stimulated deposition of elastin will hold theinjured tissue together. Increased elastin synthesis at a treatment sitemay further act as a chemotactic attractant for fibroblasts, endothelialcells, and inflammatory cells, which can promote healing of the injuredtissue. Elastin synthesis at the site of injury may also lessen scarringsince scar tissue is devoid of elastin, and elastin is an importantcomponent of uninjured skin. The stimulation and secretion of elastininto the matrix will also generally provide a favorable environment forthe cells that participate in the healing process, further accentuatingthe wound healing process.

A therapeutic composition of the invention may be injected into coronaryarteries during balloon angioplasty, injected intravenously, or injecteddirectly into myocardial post-infarct scar tissue during open heartsurgery as a means of stimulating new elastogenesis that would lendstrength and resiliency to the post-infarct scar of the human heart.

While the making and using of various embodiments of the presentinvention are discussed in detail below, it should be appreciated thatthe present invention provides many applicable inventive concepts whichcan be embodied in a wide variety of specific contexts. The specificembodiments discussed herein are merely illustrative of specific ways tomake and use the invention and do not delimit the scope of theinvention. Various modifications and combinations of the illustrativeembodiments, as well as other embodiments of the invention, will beapparent to persons skilled in the art upon reference to thedescription.

The therapeutic compositions of the present invention induce synthesisand deposition of elastin, induce cellular proliferation, and inducecellular differentiation in normal human dermal fibroblasts. Thefollowing effects in culture compositions are better understood inreference to the examples below.

EXAMPLES Example 1

Diverse topical products and injectable fillers used for correctingfacial wrinkles induce rather short-lived effects because they targetreplacement of dermal collagen and hyaluronan, matrix components oflimited biologic durability. We have tested the potential biologicaleffect of ProK on dermal fibroblasts derived from females of differentages. Northern blots, quantitative immunohistochemistry and metabolicassays were used to assess effects of ProK-60 on proliferation andmatrix production in primary cultures of dermal fibroblasts, in culturesof skin explants and after implantation of stimulated fibroblasts intothe skin of athymic nude mice.

ProK-60 increased proliferation (25-30%) of cultured dermal fibroblastsand significantly enhanced their production of new elastic fibers(>250%) and collagen fibers (100%). These effects were mostly mediatedby stimulation of cellular elastin receptor. In contrast, ProK-60inhibited production of fibronectin (˜30%) and chondroitin sulfateproteoglycans (˜50%). ProK-60 also activated proliferation of dermalfibroblasts, mostly derived from the stratum basale and induceddeposition of elastic fibers in cultures of skin explants. Moreover,human fibroblasts pre-treated with ProK-60 produced abundant elasticfibers after their injection into the skin of athymic nude mice.

Materials

All chemical grade reagents and antibody to chondroitin sulfate wereobtained from Sigma (St. Louis, Mo.). Alpha-minimum essential medium,fetal bovine serum (FBS), and other cell culture products were obtainedfrom GIBCO Life Technologies (Burlington, Ont., Canada). The antibodiesto tropoelastin, desmosine, VGVAPG and microfibrillar proteins fibrillin1, MAGP and LTBP2 were obtained from Elastin Products Co. Inc.(Owensville, Mich.). Monoclonal antibody to fibronectin (mAB1940) wasfrom Chemicon (Temecula, Calif.). Polyclonal antibodies to humancollagen type I and IV were a generous gift of Dr. Larry W. Fischer fromThe National Institute of Health (Bethesda, Md.). The 67 kDa elastinbinding protein was detected with our anti S-GAL polyclonal antibody.Antibodies recognizing growth factors, PDGF, aFGF, bFGF and IL-1b werepurchased from Genzyme (Cambridge, Mass.). Secondary antibodies,fluoresceinconjugated goat anti-rabbit (GAR-FITC) and goat anti-mouse(GAM-FITC) were purchased from Sigma. Horseradish peroxidase-conjugatedsecondary antibodies used for Western blotting were from Biorad(Hercules, Calif.). The chemiluminescence detection kit and radiolabeledreagents, [³H]-valine and [³H]-thymidine, were purchased from AmershamCanada Ltd. (Oakville, Ont., Canada).

Production of Biologically Active Peptide Preparations Derived fromElastic Fibers (Prok-60 and ProK-60P)

The starting product, obtained after neutral extraction of bovine neckligament, called Elastin E60 (Elastin Products Co., St. Louis, Mo.) wasresuspended in 50 mM Tris-HCl buffer (pH 8.5) at a ratio of 5 g ElastinE60 to 1 liter of buffer (w/v). The mixture was equilibrated to 60° C.in a water bath with shaker. Calcium Acetate (Sigma-Aldrich, St. Louis,Mo.) was added to the mixture to obtain a final concentration of 2 mM.Proteinase K (>30 units/mg protein, Product No. P5056, from Tritirachiumalbum, Sigma Co., St. Louis, Mo.) was then added to mixture at a ratioof 10 mg enzyme to 1 g elastin. Digestion was allowed to proceed for 4 hwith constant shaking. The digest was then filtered through a 10 kDacutoff tangential flow filter made from regenerated cellulose (HeliconSS50 Spiral Wound Filter Cartridge PLGC 10 kDa 4.0 mm², Product No.CDUF050LG, Millipore, Bedford, Mass.) to remove Proteinase K enzyme and≧10 kDa elastin polypeptides. The filtered soluble peptide mixture waslyophilized to a dry powder and stored at 4° C. Analysis ofchromatographic and SDS-PAGE profiles of different batches of ProK-60indicated that the above-described procedure affords production of ahighly uniform mix of peptides (data not shown). Moreover, we observedvery similar levels of induced elastogenesis in primary dermalfibroblast cultures for four ProK-60 production batches with a standarddeviation of 7%. Given these results, we assume that we can achieveroughly 93% batch reproducibility as a function of elastogenic potentialof each ProK-60 production batch.

The final peptide preparation, ProK-60 was tested alone or furtherupgraded to ProK-60P preparation when mixed with chemical preservatives(K-Sorbate 0.25%, Na-benzoate 0.25% and cetyl pyridinium chloride 0.25%(w/w)). ProK-60 was subsequently analyzed by LC-MS/MS that lead to therecovery of 111 discrete elastin peptide sequences accounting for 67% ofthe bovine elastin sequence. The concentrations of ProK-60 and ProK-60Pused herein were confirmed in pilot studies that demonstrated that 25μg/ml ProK-60 and ProK-60P induced maximal proliferation and ECMproduction (data not shown) in dermal fibroblasts.

Immuno-Characterization of ProK-60

ProK-60 was further immuno-characterized in order to determine if it wasa pure mixture of elastin derived peptides. Twenty micrograms samples ofProK-60 were separated by SDS-PAGE electrophoresis, stained withCoomassie Blue, or transferred to nitrocellulose and subjected towestern blotting with anti elastin antibody. The 20 μg samples ofProK-60 dissolved in distilled water were also directly dot-blotted witha panel of specific antibodies recognizing extracellular matrixcomponents and common growth factors. All blots were incubated with theperoxidase-conjugated secondary antibodies and visualized withchemiluminescence as previously described.

Cultures of Human Dermal Fibroblasts

Biological effects of ProK-60 and ProK-60P were tested in cultures ofskin fibroblasts derived from three healthy Caucasian females ofdifferent ages: 50, 26 and 3 years old. All fibroblasts were originallyisolated by digestion of skin biopsies with mixture of 0.25% collagenasetype I (Sigma) and 0.05% DNAse type 1 (Sigma) and then passaged bytrypsinization and maintained in alpha-minimum essential mediumsupplemented with 20 mM Hepes, 1% antibiotics and antimycotics, 1%L-glutamate and 5% fetal bovine serum (FBS). In all experiments,consecutive passages 3-7 were tested. In some experiments serum freemedium was also used.

Elastin RNA Expression

Confluent cultures of fibroblasts were maintained for 24 h in thepresence and absence of the tested reagents. Total RNA was extractedusing TRI-reagent. Steady-state levels of elastin mRNA were thenanalyzed by Northern blot hybridization using a human elastin cDNArecombinant H-11 as a probe.

Assessment of Deposition of Extracellular Matrix Components

Ten-day-old confluent cultures of fibroblasts, which produce abundantECM, were used. All cultures were fixed in cold 100% methanol at −20° C.for 30 min, blocked with 1% normal goat serum then incubated for 1 heither with 2 μg/ml of polyclonal antibodies to tropoelastin, fibrillin1, collagen type I, chondroitin sulfate and 1 μg/ml of monoclonalantibody to fibronectin. All cultures were then incubated for anadditional hour with appropriate fluorescein-conjugated secondaryantibodies (GAR-FITC or GAM-FITC). Nuclei were counterstained withpropidium iodide. Morphometric analysis of cultures immunostained withantibodies recognizing extracellular matrix components was alsoperformed using a computerized video analysis system (Image-Pro Plussoftware 3.0, Media Cybernetics, Silver Spring, Md.).

Insoluble Elastin Assay

Quadruplicate confluent cultures of dermal fibroblasts incubated for 72h in the presence and absence of the tested reagents were also exposedto 20 μCi [³H]-valine. At the end of incubation period the contents ofradioactive NaOH-insoluble elastin was assessed separately in eachculture. Final results reflecting amounts of metabolically labeled,insoluble elastin were expressed as CPM/μg DNA. DNA was determined withthe DNeasy Tissue System from Qiagen.

Assessment of Fibroblast Proliferation

Cellular proliferation rates of cultures treated with tested reagentswere assessed at the end point by counting of trypsinized cells, bytotal DNA assay, using the DNeasy Tissue System from Qiagen and byimmunochemical detection of proliferative antigen ki 67. Cellularproliferation was also assessed in parallel quadruplicate culturesexposed to [³H]-thymidine (2 MC1/well) for the last 24 h.

Since ProK-60 is a mixture of multiple peptides derived not only fromelastin, but also from other structural components of elastic fibers andsome growth factors absorbed in the ECM, we also tested whether theelastogenic and proliferative effects of the ProK-60 would be induced inthe presence of lactose, the galactosugar-containing reagentinactivating the cell surface receptor that normally interacts with theVGVAPG-like domain of elastin and transduces intracellular signals. Acontrol sugar, fucose, was also used for comparison.

Implants of Human Dermal Fibroblasts into Athymic Nude Mice

Dermal fibroblasts were isolated from skin biopsies as described above.Fibroblasts were initially cultured for 48 h in the presence and absenceof 25 μg/ml of ProK-60, then scraped, washed for 1 h in Dulbeccophosphate-buffered saline (DPBS), re-suspended in DPBS and aliquots(50,000 cells/200 μL) were injected intra-dermally into the back of 12athymic nude mice. The four left sites were injected with untreatedfibroblasts and the four right sites were injected with ProK-60-treatedfibroblasts. Additional four injections of DPBS alone composed thecontrol. Mice were sacrificed 1, 3 and 4 weeks after injections. Thehistological sections of all injected sites were stained with Movat'spentachrome. Previous, studies have confirmed that the distribution ofblack-stained material with Movat's method entirely overlaps withimmunodetectable elastin. Local IRB approval was obtained prior toinitiating animal studies.

Organ Cultures of Explants Derived from Surgical Biopsies of Human Skin

In order to further test whether ProK-60 would penetrate into skintissue and induce elastogenic effect, skin biopsies derived from fivewomen (age 28-47 years old) were cut into the small pieces and placed onthe top of metal grids immersed in the culture medium containing 5% FBSand maintained for 10 days in the presence and absence of 25 μg/ml ofProK-60 (added daily). All organ cultures were fixed in 1% bufferedformalin and their transversal serial histological sections were stainedwith Movat's pentachrome as described above. Morphometric analysis wasperformed using an Olympus AH-3 microscope attached to a CCD camera(Optronix) and a computer-generated video analysis system (Image-ProPlus software, Media Cybernetics, Silver Spring, Md.). In each analyzedexplant (three from each patient) low-power fields (1 mm²) of 50 serialsections stained with Movat's pentachrome were analyzed and allstructures stained black (elastic fibers) were counted. In eachexperimental group means±S.D. were calculated and obtained values werestatistically compared with respective controls. Informed consent andlocal IRB approval was obtained. Guidelines of the Declaration ofHelsinki for the protection of human subjects were strictly followed inconducting above surgical procedures.

In all biochemical and morphometric studies, means and standarddeviations were calculated, and statistical analyses were carried out byANOVA.

Results

Coomassie blue staining of the SDS-PAGE resolved ProK-60 preparationindicated that majority of its peptides were of molecular size rangedfrom 10 to 18 kDa. Western blots with polyclonal antielastin antibodyand with monoclonal BA4 antibody recognizing VGVAPG (SEQ ID NO. 1)domain of elastin (previously determined to elicit biological activitythrough interaction with the elastin receptor) indicated that inaddition to peptides of these molecular sizes, elastin epitopes werealso present in peptides smaller than 2 kDa. Dot immunoblots indicatedthat the ProK-60 preparation contains numerous epitopes, which wereimmunoreactive with following antibodies: polyclonal anti-tropoelastin,monoclonal BA4 recognizing VGVAPG (SEQ ID NO. 1) and other peptidesmaintaining GXXPG (SEQ ID NO. 2) sequences of elastin, polyclonalanti-desmosine and monoclonal recognizing exon 36 of elastin. Dot blotswere also immunoreactive with antibodies recognizing: fibrillin 1,MAGP1, LTBP2, versican 1, collagen type I, collagen type IV, 67 kDa EBP,PDGF, aFGF and IL-1β.

Northern blot analysis revealed that dermal fibroblasts incubated for 24h in the presence of ProK-60 had significantly up-regulated elastin mRNAlevels, as compared to their untreated counterparts, even in culturesmaintained in serum-free medium (FIG. 1A).

Results of numerous experiments involving metabolic labeling of cultureddermal fibroblasts with radioactive valine, followed by biochemicalassay of the NaOH-insoluble elastin further confirmed the stimulatoryeffect of ProK-60 and ProK-60P on the final deposition of this componentpresent only in mature elastic fibers. Both preparations were almostequally effective in stimulation of elastogenesis in fibroblasts derivedfrom individuals of different ages. The stimulatory effect of ProK-60and even better effect of ProK-60P preparation was visible inconcentrations ranging from 5 to 100 μg. Optimal stimulation, elevatingelastogenesis to levels exceeding 200% of normal values, wasconsistently achieved with a concentration 25 μg/ml of these compounds(FIG. 1B). Since deposition of [³H]-valine-labeled insoluble elastinmeasured in individual cultures (CPM/dish) was further normalized perDNA content in these cultures (CPM/1 μg DNA), our results indicate thatthe tested preparations stimulated elastogenesis in individual cells andthat a net increase was independent of mitogenic effect of thesepreparations.

Immunostainings with anti-elastin antibody further illustrated thatinsoluble elastin stimulated by ProK-60 and ProK-60P is properlydeposited in the form of extracellular elastic fibers (FIG. 1C).Morphometric analyses of immunostainings also demonstrated that ProK-60,in addition to elastin, also stimulated production and proper depositionof microfibrillar scaffold of elastic fibers marked by an increase inFibrillin 1.

Morphometry of parallel cultures immunostained with antibodiesrecognizing other components elastic fibers of extracellular matrixdemonstrated that ProK-60 also stimulated deposition of collagen type I.In contrast, cultures treated with ProK-60 demonstrated lower thannormal deposition of fibronectin and chondroitin sulfate-containingglycosaminoglycans. Results of morphometric analysis of extracellularmatrix components immunostained with the respective specific antibodiesin 10-day-old cultures of dermal fibroblasts derived from females ofdifferent ages demonstrated that both ProK-60 and ProK-60P, in additionto elastin, also stimulate production and proper deposition ofmicrofibrillar scaffold of elastic fibers marked by an increase inFibrillin 1 and deposition of collagen type I. In contrast, culturestreated with these preparations demonstrated lower than normaldeposition of fibronectin and chondroitin sulfate-containingglycosaminoglycans. In each analyzed group, 50 low-power fields (20×)from three separate cultures were analyzed and the area occupied by theparticular immunodetectable component quantified. The abundance of eachcomponent was then expressed as a percentage of the entire analyzedfield (mean±S.D.), and results from ProK-60- and ProK-60P treatedcultures were statistically compared with untreated controls.

Results of cell proliferation assays assessed either by total DNAcontent, incorporation of [³H]-thymidine or by immunodetection ofproliferative antigen Ki67, indicated that both ProK-60 and ProK-60Pinduced a slight proliferative effect that did not exceed 20-30%increase over the untreated control fibroblasts derived from all testedsubjects. This mild proliferative effect of both tested preparations wasalso visible in synchronized cultures maintained in the presence ofserum free-medium (FIG. 3). Results of total DNA assay, [³H]-thymidineincorporation and immunodetection of Ki-67 proliferative antigenconsistently demonstrated that fibroblasts treated with both testedpreparations have higher proliferation rate than the untreated controls,even when maintained in the serum free medium. In all experiments, cellswere plated with the same initial density 50,000 cells/well.Proliferation rates of ProK-60- and ProK-60P-treated fibroblasts(mean±SD) from three different experiments were statistically comparedwith untreated controls.

Results of a parallel series of experiments demonstrated that additionof lactose (reagent inactivating the cell surface elastin receptors) tocultures of human skin fibroblasts simultaneously treated with ProK-60caused significant inhibition of the proliferative and elastogeniceffects of ProK-60. Addition of fucose, sugar that does not interferewith elastin receptor, did not diminish the beneficial effects ofProK-60 (FIG. 4).

Movat's pentachrome histochemical staining of full thickness explants(derived from five different 28-47 years old females) maintained inorgan cultures for 10 days demonstrated that ProK-60 was able topenetrate into these full thickness explants and induce abundantproduction of new elastic fibers in the superficial and mid to deepdermis (FIG. 5). This observation was further endorsed by quantitativecomparison of elastic fiber content in control and ProK-60-treatedcultures (FIG. 6). Results demonstrated that dermal explants treateddaily with 25 μg/ml of ProK-60 contain significantly more elastic fibersthan explants maintained in control media. Morphometric analysis wasperformed using an Olympus AH-3 microscope attached to a CCD camera(Optronix) and a computer-generated video analysis system (Image-ProPlus software, Media Cybernetics, Silver Spring, Md.). In each analyzedexplant (three from each patient) low-power fields (1 mm²) of 50 serialsections stained with Movat's pentachrome were analyzed and allstructures stained black (elastic fibers) were counted. In eachexperimental group means±S.D. were calculated and obtained values werestatistically compared with the respective controls.

Example 2

Additional analysis (immuno-peroxidase detection of proliferativeantigen, PCNA) indicated that ProK-60 mostly stimulated proliferationand migration of cells located in the stratum basale that also producedelastic fibers (FIG. 7). Explants were maintained in organ cultures for10 days in the presence and absence of 25 μg/ml of ProK-60.Immuno-peroxidase detection (brown) of proliferative antigen, PCNA,indicates that ProK-60 mostly stimulated proliferation and migration ofcells located in the stratum basale into the superficial dermis. Movat'spentachrome stain demonstrates that fibroblasts located near thedermo-epidermal junction produce more elastic fibers (black) inProK-60-treated explants than in control counterparts (originalmagnification 400×).

Example 3

Histological examination of nude mice skin injected with ProK-60-treatedor untreated dermal fibroblasts derived from four human subjectsindicated that all 1-, 3- and 4-week-old implants were free of anyinflammatory cell infiltration and contained injected human skin cellsthat produced new extracellular matrix rich of elastic fibers (FIG. 8).In contrast to skin of control mice injected with the vehicle thatdemonstrated only few elastic fibers, all 1-, 3- and 4-week-old implantscontained injected human skin fibroblasts, which produced a newextracellular matrix, rich with elastic fibers. Injected fibroblaststhat were pre-incubated with ProK-60 produced more elastic fibers thantheir untreated counterparts. All sections were stained with Movat'spentachrome, which shows elastin as black, glycosaminoglycans as green,collagen as yellow, and cell nuclei as dark blue (original magnification400×).

Morphometric analysis of serial sections stained with Movat'spentachrome stain indicated that 4-week old implants of humanfibroblasts pre-cultured for 48 h in the presence of 25 μg/ml of ProK-60produced an average three times more elastic fibers than theirrespective untreated counterparts (data not shown).

We have demonstrated that digestion of bovine neck ligament elastin withProteinase K produces a mixture of numerous heterogenic peptides oflower molecular weight than Kappa-elastin and other chemical digests ofinsoluble elastin that have been previously described as inducers ofdiverse biological effects in cultures of several cell types, includingfibroblasts. Results indicate that our preparation, in addition to amodest (up to 30%) net increase in cellular proliferation, tremendouslyenhanced synthesis and deposition of both major components of elasticfibers, elastin and microfibrillar proteins (e.g. Fibrillin 1). Weadditionally observed increased expression of collagen type I.Interestingly, ProK-60 and ProK-60P down-regulated deposition ofchondroitin sulfate proteoglycans and fibronectin in all testedcultures. Under normal circumstances, chondroitin sulfate moietiesassociate with microfibrillar glycoproteins and play an important rolein final assembly of secreted tropoelastin on the microfibrillarscaffold. They coordinate a proper release of tropoelastin moleculesfrom their 67 kDa molecular chaperone, elastin binding protein (EBP). Ithas been shown, however, that an excess of chondroitin sulfate-bearingmoieties (e.g. versican, biglycan) accumulating in skin of patients withheritable disorder, Costello syndrome or in photo-damaged skin may causepremature release of tropoelastin molecules, thereby preventing theirnormal assembly into elastic fibers and promoting deposition ofamorphous tropoelastin coacervates that attract lipids and calciumdeposits (e.g. elastotic material). On the other hand, fibronectin,often localized on edges of individual elastic fibers, has beenimplicated as a factor limiting their thickness or a factor facilitatingECM proteolysis. Limitation of chondroitin sulfate and fibronectindeposition in response to ProK-60 ingredients may further facilitateproper assembly of normal, thick elastic fibers.

While not wishing to be bound by theory, because both proliferative andelastogenic effects of ProK-60 were significantly inhibited with lactose(reagent causing shedding of the cell surface elastin receptor), webelieve that elastin-derived peptides, comprising the bulk of ProK-60,are mostly responsible for the induction of these biological effects.Synthetic peptides containing one or two elastin derived VGVAPG (SEQ IDNO. 1) repeats (ligand for the cell-surface elastin receptor) stimulatedproliferation of dermal fibroblasts and new elastogenesis similar tothat induced by ProK-60, when tested in 20 times smaller concentrations.

Since ProK-60 also contains immunodetectable traces of other ECMmolecules and growth factors that might potentially induce biologicaleffect(s) through interaction with respective cell surface receptors,some fraction of the recorded biological effect may be induced bynon-elastin peptides.

Previous studies with human dermal fibroblasts injected into human skinand skin of athymic nude mice failed to show deposition of elasticfibers at implantation sites. Results of our experiments demonstratedthat ProK-60-pre-treated, fully differentiated human dermal fibroblastsinjected into athymic nude mice skin produced abundant extracellularmatrix, particularly rich of elastic fibers during the first week afterinjection. Noteworthy, control fibroblasts pre-cultured in mediacontaining 10% serum produced much lower synthetic ability and goodelastogenesis was not detected before 4 weeks after injection. Theseresults are of particular importance and support a novel therapeuticconcept in which short in vitro pre-treatment of biopsy-derived, fullydifferentiated dermal fibroblasts with elastogenic peptides or extractscan “jump-start” their elastogenic potential and “rejuvenate” theirphenotype after autologous implantation into wrinkles, deep lines andstretch marks.

Particularly encouraging, from the future therapeutic application pointof view, is that a certain fraction of the tested bovine digest (likelypeptides <1000 Da) induced beneficial effects in full thickness dermalexplants maintained in organ culture. ProK-60 activated cells in thestratum basale and stimulated deposition of new elastic fibersthroughout the dermis.

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontain within this specification.

1. A therapeutic composition for stimulating elastogenesis or theappearance thereof comprising a therapeutically effective amount of apopulation of cultured dermal fibroblasts, wherein said population ofcultured dermal fibroblasts is pretreated with an elastin peptidefragment, wherein said elastin peptide fragment consists of a peptidehaving the sequence selected from the group consisting of PGGVLPG (SEQID NO: 47), VGVVPG (SEQ ID NO: 48), IGLGPGGV (SEQ ID NO: 49), VGAMPG(SEQ ID NO: 51), VGLSPG (SEQ ID NO: 52), IGAMPG (SEQ ID NO: 54), IGVAPG(SEQ ID NO: 55), IGLSPG (SEQ ID NO: 56), VGAMPGAAAAAVGAMPG (SEQ ID NO:58), VGLSPGAAAAAVGLSPG (SEQ ID NO; 59), VGVAPGAAAAAVGVAPG (SEQ ID NO:60)JGAMPGAAAAAIGAMPG (SEQ ID NO: 61), IGLSPGAAAAAIGLSPG (SEQ ID NO: 63),IGVAPGAAAAAIGVAPG (SEQ ID NO: 62), and GXXPG (SEQ ID NO: 2).
 2. Thetherapeutic composition of claim 1 wherein said population of cultureddermal fibroblasts is derived from a skin biopsy.
 3. The therapeuticcomposition of claim 2 wherein said skin biopsy comprises cells of astratum basal layer.
 4. The therapeutic composition of claim 1, whereinsaid elastin peptide fragment consists of a peptide having the sequenceselected from PGGVLPG (SEQ ID NO: 47′). VGVVPG (SEQ ID NO: 48′).IGLGPGGV (SEQ ID NO: 49), and GXXPG (SEQ ID NO: 2).
 5. The therapeuticcomposition of claim 1, wherein said elastin peptide fragment consistsof a peptide having the sequence selected from PGGVLPG (SEQ ID NO: 47′).VGVVPG (SEQ ID NO: 48). IGLGPGGV (SEQ ID NO: 49′), and GXXPG (SEQ ID NO:2).
 6. The therapeutic composition of claim 1 wherein said elastinpeptide fragment is derived from a plant.
 7. The therapeutic compositionof claim 1 wherein said elastin peptide fragment is derived from ricebran.